Approach 1. A region of the zebrafish embryos yolk, the yolk sycytial layer (YSL) contains essential signals for mesoderm and endoderm induction. We devised a molecular strategy for isolating genes expressed in this region. We developed a bioinformatic method for validating our molecular strategy that will be of broad use to zebrafish researchers. We also characterized the expression of 68 genes implicated by this stategy, identifying YSL expression for ten genes (published in 2010). We tested these ten genes and found a master regulator transcription factor, Mxtx2, that participates in the generation of YSL signals. Our research program this year has focused on the functions of Mxtx2. We determined that Mxtx2 initiates YSL transcription of the nodal-related 2 (ndr2) gene, which together with ndr1 is required for much of mesoderm and endoderm differentiation. We uncovered a combination of non-embryonic sources of nodal-related ligands that account for the nodal-related portion of mesoderm and endoderm formation, namely Ndr2 and Ndr1 in the YSL and maternally-derived Ndr1. We also found that Mxtx2 acts upstream of a nodal-related gene-independent inducer of posterior mesoderm (manuscript submitted). Previously we identified risk factors for the zebrafish form of holoprosencephaly. Background: ndr2 mutant embryos have a more limited mesoderm and endoderm deficit than ndr1;ndr2 double-mutant embryos, namely a reduction in anterior axial mesoderm and endoderm, leading to holoprosencephaly (HPE). This phenotype is seen in only a fraction of ndr1 mutants, with many appearing wild type. At NHGRI, we examined the basis of sqt phenotypic variability and found environmental and genetic factors that influence HPE incidence. We extended our analysis to a novel sqt phenotype - bifurcation at the midline and find that the same genetic and environmental risk factors for HPE increase increase midline bifurcation, but that perturbations in WNT signaling specifically increase midline bifurcation incidence without affecting HPE (published in 2009). Approach 2. Identifying roles of RhoGTPase signaling in gastrulation movements. Background: RhoGEF proteins are positive regulators of RhoGTPases, which have profound roles in cellular movement and morphology. To identify RhoGEFs with roles in directing the morphogenetic events of gastrulation, we performed a loss-of-function screen. We identified 48 RhoGEFs expressed during early embryogenesis and determined the loss-of-function phenotypes for 23 of these, using a non-invasive embryo holding system we designed that allows for the parallel time-lapse documention of 54 embryos. We thus identified five RhoGEFs for which two independent MOs produced the same phenotype. Three of these, homologues of ARHGEF16, Frabin and Net1, respectively, disrupted epiboly. Two others, ARHGEF10 and PLEKHG4 homologues, caused post-gastrulation defects during somitogenesis stages. We have demonstrated the specificity of the Frabin and Net1 MOs by performing mRNA rescue experiments. A manuscript describing this work is being prepared. Approach 3. Defining roles and interactions of transcription factors in the early mesoderm and endoderm as well as transcription factors in the adjacent yolk that control key signals, nutritive and morphogenetic functions of the yolk. Background: A precise separation of newly-specified germ layer precursors in zebrafish embryos, has been untenable using conventional tools. Over previous funding years we have developed and refined a novel technique for precisely microdissecting embryonic regions of interest: FACS-assisted microdissection of photolabeled cells (FAM-P). We used this method to separate mesoderm and endoderm (mesendoderm) precursor cells from ectoderm precursor cells of pre-gastrula stage embryos, and used microarrays to assess their respective transcriptomes. We are focusing on transcription factors enriched in the mesoderm and endoderm precursors. We created a loss-of-function mutant for one such transcription factor, using the emerging zinc-finger nuclease technology, and we have evidence that this mutation affects anterior patterning via interaction with the WNT pathway. In a parallel approach, we are preparing for a comprehensive assessment of mutual regulatory interactions between multiple transcription factors enriched in mesoderm and endoderm precursors as well as in the YSL.